The Recombination Protein RAD52 Cooperates with the Excision Repair Protein OGG1 for the Repair of Oxidative Lesions in Mammalian Cells

Oxidized bases are common types of DNA modifications. Their accumulation in the genome is linked to aging and degenerative diseases. These modifications are commonly repaired by the base excision repair (BER) pathway. Oxoguanine DNA glycosylase (OGG1) initiates BER of oxidized purine bases. A small number of protein interactions have been identified for OGG1, while very few appear to have functional consequences. We report here that OGG1 interacts with the recombination protein RAD52 in vitro and in vivo. This interaction has reciprocal functional consequences as OGG1 inhibits RAD52 catalytic activities and RAD52 stimulates OGG1 incision activity, likely increasing its turnover rate. RAD52 colocalizes with OGG1 after oxidative stress to cultured cells, but not after the direct induction of double-strand breaks by ionizing radiation. Human cells depleted of RAD52 via small interfering RNA knockdown, and mouse cells lacking the protein via gene knockout showed increased sensitivity to oxidative stress. Moreover, cells depleted of RAD52 show higher accumulation of oxidized bases in their genome than cells with normal levels of RAD52. Our results indicate that RAD52 cooperates with OGG1 to repair oxidative DNA damage and enhances the cellular resistance to oxidative stress. Our observations suggest a coordinated action between these proteins that may be relevant when oxidative lesions positioned close to strand breaks impose a hindrance to RAD52 catalytic activities.

Accumulation of (5 ` S)-8,5 `-cyclo-2 `-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice

Cockayne syndrome (CS) is a human genetic disorder characterized by sensitivity to UV radiation, neurodegeneration, premature aging among other phenotypes. CS complementation group B (CS-B) gene (csb) encodes the CSB protein (CSB) that is involved in base excision repair of a number of oxidatively induced lesions in genomic DNA in vivo. We hypothesized that CSB may also play a role in cellular repair of the DNA helix-distorting tandem lesion (5`S)-8,5`-cyclo-2`-deoxyadenosine (S-cdA). Among many DNA lesions. S-cdA is unique in that it represents a concomitant damage to both the sugar and base moieties of the same nucleoside. Because of the presence of the C8-C5` covalent bond, S-cdA is repaired by nucleotide excision repair unlike most of other oxidatively induced lesions in DNA, which are subject to base excision repair. To test our hypothesis, we isolated genomic DNA from brain, kidney and liver of wild type and csb knockout (csb(-/-)) mice. Animals were not exposed to any exogenous oxidative stress before the experiment. DNA samples were analysed by liquid chromatography/mass spectrometry with isotope-dilution. Statistically greater background levels of S-cdA were observed in all three organs of csb(-/-) mice than in those of wild type mice. These results suggest the in vivo accumulation of S-cdA in genomic DNA due to lack of its repair in csb(-/-) mice. Thus, this study provides, for the first time, the evidence that CSB plays a role in the repair of the DNA helix-distorting tandem lesion S-cdA. Accumulation of unrepaired S-cdA in vivo may contribute to the pathology associated with CS. Published by Elsevier B.V.

Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1(-/-) mice compared with OGG1(+/+) mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 680-692; doi:10.1038/jcbfm.2010.147; published online 25 August 2010

Base Excision Repair Activities Differ in Human Lung Cancer Cells and Corresponding Normal Controls

Oxidative damage to DNA is thought to play a role in carcinogenesis by causing Mutations, and indeed accumulation of oxidized DNA bases has been observed in samples obtained from tumors but not from surrounding tissue within the same patient. Base excision repair (BER) is the main pathway for the repair of oxidized modifications both in nuclear and mitochondrial, DNA. In order to ascertain whether diminished BER capacity might account for increased levels of oxidative DNA damage in cancer cells, the activities of BER enzymes in three different lung cancer cell lines and their non-cancerous counterparts were measured using oligonucleotide substrates with single DNA lesions to assess specific BER enzymes. The activities of four BER enzymes, OGG1, NTH1, UDG and APE1, were compared in mitochondrial and nuclear extracts. For each specific lesion, the repair activities were similar among the three cell lines used. However, the specific activities and cancer versus control comparison differed significantly between the nuclear and mitochondrial compartments. OGG1 activity, as measured by 8-oxodA incision, was upregulated in cancer cell mitochondria but down-regulated in the nucleus when compared to control cells. Similarly, NTH1 activity was also up-regulated in mitochondrial extracts from cancer cells but did not change significantly in the nucleus. Together, these results support the idea that alterations in BER capacity are associated with carcinogenesis.

The mitochondrial transcription factor A functions in mitochondrial base excision repair

Mitochondrial transcription factor A (TFAM) is an essential component of mitochondrial nucleoids TFAM plays an important role in mitochondrial transcription and replication TFAM has been previously reported to inhibit nucleotide excision repair (NER) in vitro but NER has not yet been detected in mitochondria, whereas base excision repair (BER) has been comprehensively characterized in these organelles The BER proteins are associated with the inner membrane in mitochondria and thus with the mitochondrial nucleoid, where TFAM is also situated However, a function for TFAM in BER has not yet been investigated This study examines the role of TFAM in BER In vitro studies with purified recombinant TFAM indicate that it preferentially binds to DNA containing 8-oxoguanines, but not to abasic sites, uracils, or a gap in the sequence TFAM inhibited the in vitro incision activity of 8-oxoguanine DNA glycosylase (OGG1), uracil-DNA glycosylase (UDG), apurinic endonuclease 1 (APE1), and nucleotide incorporation by DNA polymerase gamma (pol gamma) On the other hand, a DNA binding-defective TFAM mutant, L58A, showed less inhibition of BER in vitro Characterization of TFAM knockdown (KD) cells revealed that these lysates had higher 8oxoG incision activity without changes in alpha OGG1 protein levels TFAM KD cells had mild resistance to menadione and increased damage accumulation in the mtDNA when compared to the control cells In addition, we found that the tumor suppressor p53, which has been shown to interact with and alter the DNA binding activity of TFAM, alleviates TFAM-Induced inhibition of BER proteins Together, the results suggest that TFAM modulates BER in mitochondria by virtue of its DNA binding activity and protein interactions Published by Elsevier B V

Predictive validity and classification accuracy of ActiGraph energy expenditure equations and cut-points in young children

Objectives
Evaluate the predictive validity of ActiGraph energy expenditure equations and the classification accuracy of physical activity intensity cut-points in preschoolers.

Methods
Forty children aged 4–6 years (5.3±1.0 years) completed a ~150-min room calorimeter protocol involving age-appropriate sedentary, light and moderate-to vigorous-intensity physical activities. Children wore an ActiGraph GT3X on the right mid-axillary line of the hip. Energy expenditure measured by room calorimetry and physical activity intensity classified using direct observation were the criterion methods. Energy expenditure was predicted using Pate and Puyau equations. Physical activity intensity was classified using Evenson, Sirard, Van Cauwenberghe, Pate, Puyau, and Reilly, ActiGraph cut-points.

Results
The Pate equation significantly overestimated VO2 during sedentary behaviors, light physical activities and total VO2 (P<0.001). No difference was found between measured and predicted VO2 during moderate-to vigorous-intensity physical activities (P = 0.072). The Puyau equation significantly underestimated activity energy expenditure during moderate-to vigorous-intensity physical activities, light-intensity physical activities and total activity energy expenditure (P<0.0125). However, no overestimation of activity energy expenditure during sedentary behavior was found. The Evenson cut-point demonstrated significantly higher accuracy for classifying sedentary behaviors and light-intensity physical activities than others. Classification accuracy for moderate-to vigorous-intensity physical activities was significantly higher for Pate than others.

Conclusion
Available ActiGraph equations do not provide accurate estimates of energy expenditure across physical activity intensities in preschoolers. Cut-points of ≤25counts⋅15 s−1 and ≥420 counts⋅15 s−1 for classifying sedentary behaviors and moderate-to vigorous-intensity physical activities, respectively, are recommended.

Validation and calibration of the activPAL™ for estimating METs and physical activity in 4-6 year olds

Examine the predictive validity of the activPAL™ metabolic equivalents equation, develop an activPAL™ threshold value to define moderate-to vigorous-intensity physical activities, and examine the classification accuracy of the developed moderate-to vigorous-intensity physical activities threshold value in 4- to 6-year-old children.

Evaluation of Actical equations and thresholds to predict physical activity intensity in young children

This study examined the validity of current Actical activity energy expenditure (AEE) equations and intensity cut-points in preschoolers using AEE and direct observation as criterion measures. Forty 4–6-year-olds (5.3 ± 1.0 years) completed a ~150-min room calorimeter protocol involving age-appropriate sedentary behaviours (SBs), light intensity physical activities (LPAs) and moderate-to-vigorous intensity physical activities (MVPAs). AEE and/or physical activity intensity were calculated using Actical equations and cut-points by Adolph, Evenson, Pfeiffer and Puyau. Predictive validity was examined using paired sample t-tests. Classification accuracy was evaluated using weighted kappas, sensitivity, specificity and area under the receiver operating characteristic curve. The Pfeiffer equation significantly overestimated AEE during SB and underestimated AEE during LPA (P < 0.0125 for both). There was no significant difference between measured and predicted AEEs during MVPA. The Adolph cut-point showed significantly higher accuracy for classifying SB, LPA and MVPA than all others. The available Actical equation does not provide accurate estimates of AEE across all intensities in preschoolers. However, the Pfeiffer equation performed reasonably well for MVPA. Using cut-points of ≤6 counts · 15 s−1, 7–286 counts · 15 s−1 and ≥ 287 counts · 15 s−1 when classifying SB, LPA and MVPA, respectively, is recommended.

Objectively measured physical activity and sedentary time in youth: The International children's accelerometry database (ICAD)

Background: Physical activity and sedentary behaviour in youth have been reported to vary by sex, age, weight status and country. However, supporting data are often self-reported and/or do not encompass a wide range of ages or geographical locations. This study aimed to describe objectively-measured physical activity and sedentary time patterns in youth. Methods: The International Children's Accelerometry Database (ICAD) consists of ActiGraph accelerometer data from 20 studies in ten countries, processed using common data reduction procedures. Analyses were conducted on 27,637 participants (2.8-18.4 years) who provided at least three days of valid accelerometer data. Linear regression was used to examine associations between age, sex, weight status, country and physical activity outcomes. Results: Boys were less sedentary and more active than girls at all ages. After 5 years of age there was an average cross-sectional decrease of 4.2 % in total physical activity with each additional year of age, due mainly to lower levels of light-intensity physical activity and greater time spent sedentary. Physical activity did not differ by weight status in the youngest children, but from age seven onwards, overweight/obese participants were less active than their normal weight counterparts. Physical activity varied between samples from different countries, with a 15-20 % difference between the highest and lowest countries at age 9-10 and a 26-28 % difference at age 12-13. Conclusions: Physical activity differed between samples from different countries, but the associations between demographic characteristics and physical activity were consistently observed. Further research is needed to explore environmental and sociocultural explanations for these differences.

1950 En torno al Museo Louisiana 1970

La tesis “1950 En torno al Museo Louisiana 1970” analiza varias obras relacionadas con el espacio doméstico, que se realizaron entre 1950 y 1970 en Dinamarca, un periodo de esplendor de la Arquitectura Moderna. Tras el aislamiento y restricciones del conflicto bélico que asoló Europa, los jóvenes arquitectos daneses, estaban deseosos por experimentar nuevas ideas de procedencia internacional, favorecidos por diferentes circunstancias, encuentran el mejor campo de ensayo en el espacio doméstico. La mejor arquitectura doméstica en Dinamarca, de aquel periodo, debe entenderse como un sistema compuesto por diferentes autores, que tienen en común muchas más similitudes que diferencias, se complementan unos a otros. Para la comprensión y el entendimiento de ello se hace necesario el estudio de varias figuras y edificios, que completen este sistema cuya investigación está escasamente desarrollada. La tesis propone un viaje para conocer los nombres de algunos de sus protagonistas, que mostraron con su trabajo, que tradición y vanguardia no estarán reñidas. El objetivo es desvelar las claves de la Modernidad Danesa, reconocer, descubrir y recuperar el legado de algunos de sus protagonistas en el ámbito doméstico, cuya lección se considera de total actualidad. Una arquitectura que asume las aportaciones extranjeras con moderación y crítica, cuya íntima relación con la tradición arquitectónica y la artesanía propias, será una de sus notas especiales. Del estudio contrastado de varios proyectos y versiones, se obtienen valores comunes entre sus autores, al igual que se descubren sus afinidades o diferencias respecto a los mismos asuntos; que permitirán comprender sus actuaciones según las referencias e influencias, y definir las variables que configuran sus espacios arquitectónicos. La línea de conexión entre los edificios elegidos será su particular relación con la naturaleza y el lugar en que se integran. La fachada, lugar donde se negociará la relación entre el interior y el paisaje, será un elemento entendido de un modo diferente en cada uno de ellos, una relación que se extenderá en todas ellas, más allá de su perímetro. La investigación se ha estructurado en seis capítulos, que van precedidos de una Introducción. En el capítulo primero, se estudian y se señalan los antecedentes, las figuras y edificios más relevantes de la Tradición Danesa, para la comprensión y el esclarecimiento de algunas de las claves de su Modernidad en el campo de la Arquitectura, que se produce con una clara intención de encontrar su propia identidad y expresión. Esta Modernidad floreciente se caracteriza por la asimilación de otras culturas extranjeras desde la moderación, con un punto de vista crítico, y encuentra sus raíces ancladas a la tradición arquitectónica y la artesanía propia, que fragua en la aparición de un ideal común con enorme personalidad y que hoy se valora como una auténtica aportación de una cultura considerada entonces periférica. Se mostrará el debate y el camino seguido por las generaciones anteriores, a las obras análizadas. Las sensibilidades por lo vernáculo y lo clásico, que aparentemente son contradictorias, dominaran el debate con la misma veracidad y respetabilidad. La llamada tercera generación por Sigfried Giedion reanudará la práctica entre lo clásico y lo vernáculo, apoyados en el espíritu del trabajo artesanal y de la tradición, con el objetivo de conocer del acto arquitectónico su “la verdad” y “la esencia original”. El capítulo segundo, analiza la casa Varming, de 1953, situada en un área residencial de Gentofte, por Eva y Nils Koppel, que reinterpreta la visión de Asplund de un paisaje interior continuación del exterior, donde rompen la caja de ladrillo macizo convencional propia de los años 30. Es el ejemplo más poderoso de la unión de tradición e innovación en su obra residencial. Sus formas sobrias entre el Funcionalismo Danés y la Modernidad se singularizarán por su abstracción y volúmenes limpios que acentúan el efecto de su geometría, prismática y brutalista. El desplazamiento de los cuerpos que lo componen, unos sobre otros, generan un ritmo, que se producirá a otras escalas, ello unido a las variaciones de sus formas y a la elección de sus materiales, ladrillo y madera, le confieren a la casa un carácter orgánico. El edificio se ancla a la tierra resolviéndose en diferentes niveles tras el estudio del lugar y su topografía. El resultado es una versión construida del paisaje, en la cual el edificio da forma al lugar y ensalza la experiencia del escenario natural. La unidad de las estructuras primitivas, parece estar presente. Constituye un ejemplo de la “La idea de Promenade de Asplund”, el proyecto ofrece diferentes recorridos, permitiendo su propia vivencia de la casa, que ofrece la posibilidad vital de decidir. El capítulo tercero trata sobre el pabellón de invitados de Niels Bohr de 1957, situado un área boscosa, en Tisvilde Hegn, fue el primer edificio del arquitecto danés Vilhelm Wohlert. Arraigado a la Tradición Danesa, representa una renovación basada en la absorción de influencias extranjeras: la Arquitectura Americana y la Tradición Japonesa. La caja de madera, posada sobre un terreno horizontal, tiene el carácter sensible de un organismo vivo, siempre cambiante según las variaciones de luz del día o temperatura. Cuando se abre, crea una prolongación del espacio interior, que se extiende a la naturaleza circundante, y se expande hacia el espacio exterior, permitiendo su movilización. Se establece una arquitectura de flujos. Hay un interés por la materia, su textura y el efecto emocional que emana. Las proporciones y dimensiones del edificio están reguladas por un módulo, que se ajusta a la medida del hombre, destacando la gran unidad del edificio. La llave se su efecto estético está en su armonía y equilibrio, que transmiten serenidad y belleza. El encuentro con la naturaleza es la lección más básica del proyecto, donde un mundo de relaciones es amable al ser humano. El capítulo cuarto, analiza el proyecto del Museo Louisiana de 1958, en Humlebæk, primer proyecto de la pareja de arquitectos daneses Jørgen Bo y Vilhelm Wohlert. La experiencia en California de Wohlert donde será visitado por Bo, será trascendental para el desarrollo de Louisiana, donde la identidad Danesa se fusiona con la asimilación de otras culturas, la arquitectura de Frank Lloyd Wright, la del área de la Bahía y la Tradición Japonesa principalmente. La idea del proyecto es la de una obra de arte integral: arquitectura, arte y paisaje, coexistirían en un mismo lugar. Diferentes recursos realzarán su carácter residencial, como el uso de los materiales propios de un entorno doméstico, la realización a la escala del hombre, el modo de usar la iluminación. Cubiertas planas que muestran su artificialidad, parecen flotar sobre galerías acristaladas, acentuarán la fuerza del plano horizontal y establecerán un recorrido en zig-zag de marcado ritmo acompasado. Ritmo que tiene que ver con la encarnación del pulso de la naturaleza, que se acompaña de juegos de luz, y de otras vibraciones materiales a diferentes escalas, imagen, que encuentra una analogía semejante en la cultura japonesa. Todo se coordina con la trama estructural, que conlleva a una construcción y proporción disciplinada. Louisiana atiende al principio de crecimiento de la naturaleza, con la que su conexión es profunda. Hay un dinamismo expresado por el despliegue del edificio, que evoca a algunos proyectos de la Tradición Japonesa. Los blancos muros tienen su propia identidad como formas en sí mismas, avanzan prolongándose fuera de la línea del vidrio, se mueven libremente siguiendo el orden estructural, acompañando al espacio que fluye, en contacto directo con la naturaleza que está en un continuo estado de flujos. Se da todo un mundo de relaciones, donde existe un dialogo entre el paisaje, arte y arquitectura. El capítulo quinto, se dedica a analizar la segunda casa del arquitecto danés Halldor Gunnløgsson, de 1959. Evoca a la Arquitectura Japonesa y Americana, pero es principalmente resultado de una fuerte voluntad y disciplina artística personal. La cubierta, plana, suspendida sobre una gran plataforma pavimentada, que continúa la sección del terreno construyendo de lugar, tiene una gran presencia y arroja una profunda sombra bajo ella. En el interior un espacio único, que se puede dividir eventualmente, discurre en torno a un cuerpo central. El espacio libre fluye, extendiéndose a través de la transparencia de sus ventanales a dos espacios contrapuestos: un patio ajardinado íntimo, que inspira calma y sosiego; y la naturaleza salvaje del mar que proyecta el color del cielo, ambos en constante estado de cambio. El proyecto se elabora de un modo rigurosamente formal, existiendo al mismo tiempo un perfecto equilibrio entre la abstracción de su estructura y su programa. La estructura de madera cuyo orden se extiende más allá de los límites de su perímetro, está formada por pórticos completos como elementos libres, queda expuesta, guardando una estrecha relación con el concepto de modernidad de Mies, equivalente a la arquitectura clásica. La preocupación por el efecto estético es máxima, nada es improvisado. Pero además la combinación de materiales y el juego de las texturas hay una cualidad táctil, cierto erotismo, que flota alrededor de ella. La precisión constructiva y su refinamiento se acercan a Mies. La experiencia del espacio arquitectónico es una vivencia global. La influencia de la arquitectura japonesa, es más conceptual que formal, revelada en un respeto por la naturaleza, la búsqueda del refinamiento a través de la moderación, la eliminación de los objetos innecesarios que distraen de la experiencia del lugar y la preocupación por la luz y la sombra, donde se establece cierto paralelismo con el oscuro mundo del invierno nórdico. Hay un entendimiento de que el espacio, en lugar de ser un objeto inmaterial definido por superficies materiales se entiende como interacciones dinámicas. El capítulo sexto. Propone un viaje para conocer algunas de las viviendas unifamiliares más interesantes que se construyeron en el periodo, que forman parte del sistema investigado. Del estudio comparado y orientado en varios temas, se obtienen diversa conclusiones propias del sistema estudiado. La maestría de la sustancia y la forma será una característica distintiva en Dinamarca, se demuestra que hay un acercamiento a la cultura de Oriente, conceptual y formal, y unos intereses comunes con cierta arquitectura Americana. Su lección nos sensibiliza hacia un sentido fortalecido de proporción, escala, materialidad, textura y peso, densidad del espacio, se valora lo táctil y lo visual, hay una sensibilidad hacia la naturaleza, hacia lo humano, hacia el paisaje, la integridad de la obra. ABSTRACT The thesis “1950 around the Louisiana Museum 1970” analyses several works related to domestic space, which were carried out between 1950 and 1970 in Denmark, a golden age of modern architecture. After the isolation and limitations brought about by the war that blighted Europe, young Danish architects were keen to experiment with ideas of an international origin, encouraged by different circumstances. They find the best field of rehearsal to be the domestic space. The best architecture of that period in Denmark should be understood as a system composed of different authors, who have in common with each other many more similarities than differences, thus complimenting each other. In the interests of understanding, the study of a range of figures and buildings is necessary so that this system, the research of which is still in its infancy, can be completed. The thesis proposes a journey of discovery through the names of some of those protagonists who were showcased through their work so that tradition and avant- garde could go hand in hand. The objective is to unveil the keys to Danish Modernity; to recognise, discover and revive the legacy of some of its protagonists in the domestic field whose lessons are seen as entirely of the present. For an architect, the taking on of modern contributions with both moderation and caution, with its intimate relationship with architectural tradition and its own craft, will be one of his hallmarks. With the study set against several projects and versions, one can derive common values among their authors. In the same way their affinities and differences in respect of the same issue emerge. This will allow an understanding of their measures in line with references and influences and enable the defining of the variables of their architectural spaces. The common line between the buildings selected will be their particular relationship with nature and the space with which they integrate. The façade, the place where the relationship between the interior and the landscape would be negotiated, wouldl be the discriminating element in a distinct way for each one of them. It is through each of these facades that this relationship would extend, and far beyond their physical perimeter. The investigation has been structured into six chapters, preceded by an introduction. The first chapter outlines and analyses the backgrounds, figures and buildings most relevant to the Danish Tradition. This is to facilitate the understanding and elucidation of some of the keys to its modernity in the field of architecture, which came about with the clear intention to discover its own identity and expression. This thriving modernity is characterized by its moderate assimilation with foreign cultures with a critical eye, and finds its roots anchored in architectural tradition and its own handcraft. It is forged in the emergence of a common ideal of enormous personality which today has come to be valued as an authentic contribution to the sphere from a culture that was formerly seen as on the peripheries. What will be demonstrated is the path taken by previous generations to these works and the debate that surrounds them. The sensibilities for both the vernacular and the classic, which at first glance may seem contradictory, will dominate the debate with the same veracity and respectability. The so-called third generation of Sigfried Giedion will revive the process between the classic and the vernacular, supported in spirit by the handcraft work and by tradition, with the objective of discovering the “truth” and the “original essence” of the architectural act. The second chapter analyzes the Varming house, built by Eva and Nils Koppel 1953, which is situated in a residential area of Gentofte. This reinterprets Asplund’s vision of an interior landscape extending to the exterior, where we see a break with the conventional sturdy brick shell of the 1930s. It is the most powerful example of the union of tradition and innovation in his their residential work. Their sober forms caught between Danish Functionalism and modernity are characterized by their abstraction and clean shapes which accentuate their prismatic and brutal geometry, The displacement of the parts of which they are composed, one over the other, generate a rhythm. This is produced to varying scales and is closely linked to its forms and the selection of materials – brick and wood – that confer an organic character to the house. The building is anchored to the earth, finding solution at different levels through the study of place and topography. The result is an adaption constructed out of the landscape, in which the building gives form to the place and celebrates the experience of the natural setting. The unity of primitive structures appears to be present. It constitutes an example of “Asplund’s Promenade Idea”. Different routes of exploration within are available to the visitor, allowing for one’s own personal experience of the house, allowing in turn for the vital chance to decide. The third chapter deals with Niels Bohr’s guest pavilion. Built in 1957, it is situated in a wooded area of Tisvilde Hegn and was the architect Vilhelm Wohlert’s first building. Rooted in the Danish Tradition, it represents a renewal based on the absorption of foreign influences: American architecture and the Japanese tradition. The wooden box, perched atop a horizontal terrain, possesses the sensitive character of the living organism, ever-changing in accordance with the variations in daylight and temperature. When opened up, it creates an elongation of the interior space which extends into the surrounding nature and it expands towards the exterior space, allowing for its mobilisation. It establishes an architecture of flux. There is interest in the material, its texture and the emotional effect it inspires. The building’s proportions and dimensions are regulated by a module, which is adjusted by hand, bringing out the great unity of the building. The key to its aesthetic effect is its harmony and equilibrium, which convey serenity and beauty. The meeting with nature is the most fundamental lesson of the project, where a world of relationships softens the personality of the human being. The fourth chapter analyzes the Louisiana Museum project of 1958 in 1958. It was the first project of the Danish architects Jørgen Bo and Vilhelm Wohlert. Wohlert’s experience in California where he was visited by Bo would be essential to the development of Louisiana, where the Danish identity is fused in assimilation with other cultures, the architecture of Frank Lloyd Wright, that of the Bahía area and principally the Japanese tradition. The idea of the project was for an integrated work of art: architecture, art and landscape, which would coexist in the same space. A range of different resources would realize the residential character, such as the use of materials taken from a domestic environment, the attainment of human scale and the manner in which light was used. Flat roof plans that show their artificiality and appear to float over glassed galleries. They accentuate the strength of the horizontal plan and establish a zigzag route of marked and measured rhythm. It is a rhythm that has to do with the incarnation of nature’s pulse, which is accompanied with plays of light, as well as material vibrations of different scales, imagery which uncovers a parallel analogy with Japanese culture. Everything is coordinated along a structural frame, which involves a disciplined construction and proportion. Louisiana cherishes nature’s principle of growth, to which its connection is profound. Here is a dynamism expressed through the disposition of the building, which evokes in some projects the Japanese tradition. The white walls possess their own identity as forms in their own right. They advance, extending beyond the line of glass, moving freely along the structural line, accompanying a space that flows and in direct contact with nature that is itself in a constant state of flux. It creates a world of relationships, where dialogue exists between the landscape, art and architecture. The fifth chapter is dedicated to analyzing the Danish architect Halldor Gunnløgsson’s second house, built in 1959. It evokes both Japanese and American architecture but is principally the result of a strong will and personal artistic discipline. The flat roof suspended above a large paved platform – itself continuing the constructed terrain of the place – has great presence and casts a heavy shadow beneath. In the interior, a single space, which can at length be divided and which flows around a central space. The space flows freely, extending through the transparency of its windows which give out onto two contrasting locations: an intimate garden patio, inspiring calm and tranquillity, and the wild nature of the sea which projects the colour of sky, both in a constant state of change. The project is realized in a rigorously formal manner. A perfect balance exists between the abstraction of his structure and his project. The wooden structure, whose order extends beyond the limits of its perimeter, is formed of complete porticos of free elements. It remains exposed, maintaining a close relationship with Mies’ concept of modernity, analogous to classical architecture. The preoccupation with the aesthetic effect is paramount and nothing is improvised. But in addition to this - the combination of materials and the play of textures - there is a tactile quality, a certain eroticism, which lingers all about. The constructive precision and its refinement are close to Mies. The experience of the architectural space is universal. The influence of Japanese architecture, more conceptual than formal, is revealed in a respect for nature. It can be seen in the search for refinement through moderation, the elimination of the superfluous object that distract from the experience of place and the preoccupation with light and shade, where a certain parallel with the dark world of the Nordic winter is established. There is an understanding that space, rather than being an immaterial object defined by material surfaces, extends instead as dynamic interactions. The sixth chapter. This proposes a journey to discover some of the unfamiliar residences of most interest which were constructed in the period, and which form part of the system being investigated. Through the study of comparison and one which is geared towards various themes, diverse conclusions are drawn regarding the system being researched. The expertise in substance and form will be a distinctive characteristic in Denmark, demonstrating an approach to the culture of the Orient, both conceptual and formal, and some common interests in certain American architecture. Its teachings sensitize us to a strengthened sense of proportion, scale, materiality, texture and weight and density of space. It values both the tactile and the visual. There is a sensitivity to nature, to the human, to the landscape and to the integrity of the work.

Altered spectra of hypermutation in antibodies from mice deficient for the DNA mismatch repair protein PMS2

Mutations are introduced into rearranged Ig variable genes at a frequency of 10−2 mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2−/− human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.

The C-terminal Domain of p21 Inhibits Nucleotide Excision Repair In Vitro and In Vivo

The protein p21Cip1, Waf1, Sdi1 is a potent inhibitor of cyclin-dependent kinases (CDKs). p21 can also block DNA replication through its interaction with the proliferating cell nuclear antigen (PCNA), which is an auxiliary factor for polymerase δ. PCNA is also implicated in the repair resynthesis step of nucleotide excision repair (NER). Previous studies have yielded contradictory results on whether p21 regulates NER through its interaction with PCNA. Resolution of this controversy is of interest because it would help understand how DNA repair and replication are regulated. Hence, we have investigated the effect of p21 on NER both in vitro and in vivo using purified fragments of p21 containing either the CDK-binding domain (N terminus) or the PCNA binding domain (C terminus) of the protein. In the in vitro studies, DNA repair synthesis was measured in extracts from normal human fibroblasts using plasmids damaged by UV irradiation. In the in vivo studies, we used intact and permeabilized cells. The results show that the C terminus of the p21 protein inhibits NER both in vitro and in vivo. These are the first in vivo studies in which this question has been examined, and we demonstrate that inhibition of NER by p21 is not merely an artificial in vitro effect. A 50% inhibition of in vitro NER occurred at a 50:1 molar ratio of p21 C-terminus fragment to PCNA monomer. p21 differentially regulates DNA repair and replication, with repair being much less sensitive to inhibition than replication. Our in vivo results suggest that the inhibition occurs at the resynthesis step of the repair process. It also appears that preassembly of PCNA at repair sites mitigates the inhibitory effect of p21. We further demonstrate that the inhibition of DNA repair is mediated via binding of p21 to PCNA. The N terminus of p21 had no effect on DNA repair, and the inhibition of DNA repair by the C terminus of p21 was relieved by the addition of purified PCNA protein.

The Werner Syndrome Protein Is Involved in RNA Polymerase II Transcription

Werner syndrome (WS) is a human progeroid syndrome characterized by the early onset of a large number of clinical features associated with the normal aging process. The complex molecular and cellular phenotypes of WS involve characteristic features of genomic instability and accelerated replicative senescence. The gene involved (WRN) was recently cloned, and its gene product (WRNp) was biochemically characterized as a helicase. Helicases play important roles in a variety of DNA transactions, including DNA replication, transcription, repair, and recombination. We have assessed the role of the WRN gene in transcription by analyzing the efficiency of basal transcription in WS lymphoblastoid cell lines that carry homozygous WRN mutations. Transcription was measured in permeabilized cells by [3H]UTP incorporation and in vitro by using a plasmid template containing the RNA polymerase II (RNA pol II)–dependent adenovirus major late promoter. With both of these approaches, we find that the transcription efficiency in different WS cell lines is reduced to 40–60% of the transcription in cells from normal individuals. This defect can be complemented by the addition of normal cell extracts to the chromatin of WS cells. Addition of purified wild-type WRNp but not mutated WRNp to the in vitro transcription assay markedly stimulates RNA pol II–dependent transcription carried out by nuclear extracts. A nonhelicase domain (a direct repeat of 27 amino acids) also appears to have a role in transcription enhancement, as revealed by a yeast hybrid–protein reporter assay. This is further supported by the lack of stimulation of transcription when mutant WRNp lacking this domain was added to the in vitro assay. We have thus used several approaches to show a role for WRNp in RNA pol II transcription, possibly as a transcriptional activator. A deficit in either global or regional transcription in WS cells may be a primary molecular defect responsible for the WS clinical phenotype.

The ATPase Domain but Not the Acidic Region of Cockayne Syndrome Group B Gene Product Is Essential for DNA Repair

Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways.